Oxygen-resistant hydrogenases and methods for designing and making same

ABSTRACT

The invention provides oxygen-resistant iron-hydrogenases ([Fe]-hydrogenases) for use in the production of H 2 . Methods used in the design and engineering of the oxygen-resistant [Fe]-hydrogenases are disclosed, as are the methods of transforming and culturing appropriate host cells with the oxygen-resistant [Fe]-hydrogenases. Finally, the invention provides methods for utilizing the transformed, oxygen insensitive, host cells in the bulk production of H 2  in a light catalyzed reaction having water as the reactant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 10/553,097,filed Oct. 13, 2005, which is a national stage entry of InternationalApplication No. PCT/US04/11830, filed Apr. 16, 2004, which claimspriority to U.S. Provisional Application No. 60/464,081, filed Apr. 18,2003. The contents of each application listed above are incorporated byreference in their entirety.

GOVERNMENT INTEREST

The United States Government has rights in this invention pursuant tocontract DE-AC36-08GO28308 between the United States Department ofEnergy and the Alliance for Sustainable Energy, LLC, manager andoperator of the National Renewable Energy Laboratory.

FIELD OF THE INVENTION

The present invention relates to hydrogen-production by microorganisms,for example hydrogen production by green algae. More specifically, theinvention relates to methods for designing and engineering hydrogenaseenzymes with improved oxygen resistance, and to the methods fortransforming microorganisms to express these oxygen-resistanthydrogenase enzymes for use in the production of hydrogen in an oxygencontaining environment.

BACKGROUND OF THE INVENTION

Hydrogen (H₂) is becoming an attractive alternative energy source tofossil fuels due to its clean emissions and potential for cost effectiveproduction by microorganisms. As such, microorganisms that metabolize H₂are being investigated for their potential use in H₂-production. Amicroorganism of particular interest for H₂ production is the greenalga, Chlaydomonas reinhardtii, which is able to catalyzelight-dependent, H₂ production utilizing water as a reductant. Ghiradiet al., (2000) Trends Biotech. 18(12):506-511; Melis et al., (2001)Plant Physiol. 127:740-748. The benefits of using an algal system forH₂-production include the use of renewable substrates (light and water)and its potential cost-effectiveness. Melis A, Int. J. Hyd. Energy27:1217-1228. As such, there is a great deal of interest in optimizingH₂-production by green algae to maximize the potential benefit as analternative energy source.

Chlamydomonas reinhardtii, and other like microorganisms, are able toexpress a class of H₂ metabolizing enzymes called hydrogenases. Membersof this enzyme family function in either H₂-uptake (as a means toprovide reductant for substrate oxidation) or H₂-production (as a meansto eliminate excess reducing equivalents). Characterization of varioushydrogenases from multiple organisms has identified three principlehydrogenase types, broadly classified by the chemical nature of theiractive sites: [Fe]-hydrogenase, [NiFe]-hydrogenase, and non-metallic(organic) hydrogenase. Vignais et al, (2001) FEMS Micro. Rev.25:455-501; Adams. M. W., Biochem. Biophys. Acta. 1020:115-145; Buurmanet al., (2000) FEBS Letts. 485:200-204. More particularly,[Fe]-hyrdogenase have an active site containing a [4Fe-4S]-centerbridged to a [2Fe-2S]-center (H-cluster) (Peters et al., (1998) Science282:1853-1858; Nicolet et al., (1998) Structure 7:13-23), and the[NiFe]-hydrogenase have an active site containing a [4Fe-4S]-centerbridged to a [NiFe]-center (Volbeda et al., (1995) Nature 373:580-587).Coordination of the metal prosthetic groups to the active sites is madeby cysteinyl, CN⁻, and CO ligands. Further, within each hydrogenasegroup are monomeric, or multimeric enzymes, that can be eithercytoplasmic or membrane bound within the cell. Vignais et al., Supra.

Although there are differences within the active sites between differentfamilies of hydrogenase, as well as between the subunit composition andlocalization between hydrogenase families, most, if not all studiedhydrogenases have exhibited some degree of sensitivity to inhibition byCO and O₂. Adams M. W. W; Volbeda et al., (1990) Int. J. Hyd. Energy27:1449-1461. Hydrogenase sensitivity to these inhibitors correlates tosome degree to the type of prosthetic group that forms the active site,for example, [Fe]-hydrogenase is highly sensitive to O₂. As such, forexample, the activity of [Fe]-hydrogenase in C. reinhardtii is verysensitive to O₂ during H₂-photoproduction under photosyntheticconditions. Ghirardi et al., (1997) App. Biochem. Biotech.63-65:141-151. Oxygen inhibition of [Fe]-hydrogenases is a majordrawback in the use of green alga for H₂ production.

One approach to overcoming this H₂ production limitation is to stressthe C. reinhardtii under photoheterotrophic, sulfur-deprived conditionsthat minimize O₂-photoproduction levels and result in sustainedH₂-production. However, this approach does not result in optimal yieldsand requires the use of sulfur-deprived/oxygen limited productiontechniques. Recently, CO and O₂ inhibition of hydrogenase activity inalga has been focused on the putative role of the H₂-channel. Forexample, it has been shown that the positioning of the Fe₂-atom in theenzyme's active site is directly at the active-site/H₂-channelinterphase, where it is easily accessed by either CO or O₂ diffusingthrough the channel. Lemon et al., (1999) Biochem. 38:12969-12973;Bennett et al., (2000) Biochem. 39:7455-7460. Further, a naturallyoccurring O₂-resistant [NiFe]-hydrogenase has been shown to have anarrower active site/H₂-channel interphase than the naturally occurringhydrogenase counterpart. Volbeda et al. (2002), Supra.

Against this backdrop the present invention has been developed.

SUMMARY OF THE INVENTION

The present invention provides oxygen-resistant hydrogenases for use inthe bulk production of H₂ in green algae cultures. In a preferredembodiment, homology modeling between known hydrogenases, e.g., CpI, andtarget hydrogenases, e.g., HydA1, was used to design and in silicoengineer an oxygen-resistant [Fe]-hydrogenase having a reduced diameterH₂-channel. Constructed polynucleotides that encode oxygen-resistant[Fe]-hydrogenase enzymes are used to transform target host cells whichwere subsequently used in the photoproduction of H₂. In preferredembodiments, the target host cells are C. reinhardtii. The inventionprovides a solution to the problem of H₂ production by green algae whenO₂ is present in the environment.

The present invention also provides host cells expressingoxygen-resistant [Fe]-hydrogenase. Host cells expressing theoxygen-resistant [Fe]-hydrogenase have significantly increased H₂production, in the presence of O₂, as compared to similarly treatedcells that do not express oxygen-resistant [Fe]-hydrogenase.

The present invention also provides polynucleotide molecules encodingHydA1V240W and other like oxygen-resistant hydrogenase polypeptides. Theinvention includes nucleic acid molecules that hybridize under highstringency conditions to the HydA1V240W polynucleotides (and other likeoxygen-resistant hydrogenase polynucleotides) of the present invention.The invention also includes variants and derivatives of theoxygen-resistant [Fe]-hydrogeanse polypeptides, including fusionproteins that confer a desired function. The invention also providesvectors, plasmids, expression systems, host cells and the like,containing the oxygen-resistant [Fe]-hydrogenase of the invention.

These and various other features and advantages of the invention will beapparent from a reading of the following detailed description and areview of the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a protein alignment using PILEUP/GENEDOC program. Aminoacid residues highlighted in black represent identities between at least5 of the iron-hydrogenases, and those highlighted in grey showsimilarity between at least 5 of the sequences (1A). FIG. 1B shows atheoretical structure of HydA1 using homology modeling to the solvedX-ray structure of CpI. The left panel shows an overlay of HydA1 andCpI, with locations of the H₂-channels and the active sites, while theright panel shows the HydA1 structure.

FIG. 2 shows the protein sequence of HydA1 aligned to the catalytic coreregion of CpI. The sequences that form the H₂-channel domain are shadedeither gray (similar) or black (identical).

FIGS. 3A and 3B show how mutations made to the HydA1H₂-channel result inpredicted H₂-channel structures. Wild type HydA1 H2-channel structure isshown in 3B, while mutant H2-channel structures are shown in 3A. Notethat for reference purposes, the channel has been divided into fourzones (black line numbered 1-4).

FIGS. 4A and 4B show side-orientation views from the active site (left)to the protein surface (right) of the H₂-channel of the wild type HydA1(4A) and mutant HydA1V240W (4B).

FIG. 5 illustrates PCR products from C. reinhardtii HydA1V240Wtransformants mt18 and mt28. Genomic DNA isolated from cc849 (wildtype), mt18, and mt28 were digested with either SacI (lanes 1-3) orEcoRI (lanes 4-6) and used as template in a PCR reaction with HydA1specific primers. Lanes 1 and 4 are wild type, lanes 2 and 5 are mt18and lanes 3 and 6 are mt28. Note that lane 7 is a DNA size marker andlane 8 is a pAIExBle control. The upper band in the stained agarose gelcorresponds to the HydA1 genomic copy, and the lower band corresponds tothe HydA1 cDNA insert.

FIG. 6 illustrates hydrogenase activity as measured by the rate of H₂evolved (μmol H₂/mg chl⁻¹/h⁻¹) under variant O₂ concentrations (0 to3.5% final O₂ concentration) and plotted relative to the activity valueobtained under completely anaerobic conditions.

FIG. 7 shows the activity of O₂-resistant [Fe]-hydrogenase as measuredin a reduced MV assay. Samples of induced cells were taken and assayedfor hydrogenase activity following exposure to various levels of O₂(0-4% final O₂ concentration). Note that hydrogenase activity wasmeasured as the rate of H2 evolved (μmol H₂/mg chl⁻¹/h⁻¹) over a30-minute incubation period and plotted relative to the activity valueobtained under completely anaerobic conditions.

FIG. 8 shows a plasmid map for the plasmid pLam91-1.

FIG. 9 shows a plasmid map for the plasmid pA1ExBle.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding ofcertain terms used herein and are not meant to limit the scope of thepresent disclosure.

“Amino acid” or “residues” refers to any of the twenty naturallyoccurring amino acids as well as any modified amino acid sequences.Modifications may include natural processes such as posttranslationalprocessing, or may include chemical modifications which are known in theart. Modifications include but are not limited to: phosphorylation,ubiquitination, acetylation, amidation, glycosylation, covalentattachment of flavin, ADP-ribosylation, cross-linking, iodination,methylation, and alike. Amino acid residue characterization can be foundin numerous citations, for example Stryer, 1995, Biochemistry,throughout the text and 17-44.

“Expression” refers to transcription and translation occurring within ahost cell. The level of expression of a DNA molecule in a host cell maybe determined on the basis of either the amount or corresponding mRNAthat is present within the cell or the amount of DNA molecule encodedprotein produced by the host cell (Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, 18.1-18.88).

“Genetically engineered” refers to any recombinant DNA or RNA methodused to create a host cell that expresses a target protein at elevatedlevels, at lowered levels, or in a mutated form. Typically, the hostcell has been transfected, transformed, or transduced with a recombinantpolynucleotide molecule, and thereby been altered so as to cause thecell to alter expression of the desired protein. Methods for geneticallyengineering host cells are well known in the art. (See Current Protocolsin Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York,1988, and quarterly updates)). Genetically engineering techniquesinclude but are not limited to expression vectors, targeted homologousrecombination and gene activation (see, for example U.S. Pat. No.5,272,071 to Chappel) and trans activation by engineered transcriptionfactors (See Segal et al., 1999, Proc Natl Acad Sci USA 96(6):2758-63).

“Hybridization” refers to the pairing of complementary polynucleotidesduring an annealing process. The strength of hybridization between twopolynucleotide molecules is impacted by the homology between the twomolecules, stringency of the conditions involved, and meltingtemperatures of the formed hybrid and the G:C ratio within thepolynucleotide. For purposes of the present invention stringencyhybridization conditions refers to the temperature, ionic strength,solvents, etc, under which hybridization between polynucleotides occurs.

“Identity” refers to a comparison between pairs of nucleic acid or aminoacid molecules. Methods for determining sequence identity are known inthe art. For example, computer programs have been developed to performthe comparison, such as the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), that uses the algorithm of Smith andWaterman (1981) Adv Appl Math 2:482-489.

“Isolating” refers to a process for separating a nucleic acid orpolypeptides from at least one contaminant with which it is normallyassociated. In preferred embodiments, isolating refers to separating anucleic acid or polypeptide from at least 50% of the contaminants withwhich it is normally associated, and more preferably from at least 75%of the contaminants with which it is normally associated.

The term “nucleic acid” refers to a linear sequence of nucleotides. Thenucleotides are either a linear sequence of polyribonucleotides orpolydeoxyribonucleotides, or a mixture of both. Examples of nucleic acidin the context of the present invention include—single and doublestranded DNA, single and double stranded RNA, and hybrid molecules thathave mixtures of single and double stranded DNA and RNA. Further, thenucleic acids of the present invention may have one or more modifiednucleotides.

The term “PCR” or “polymerase chain reaction” refers to the process toamplify nucleic acids as described in U.S. Pat. Nos. 4,683,105 and4,683,202, both owned by Roche Molecular.

“Host cell” refers to cells containing a target nucleic acid molecule,for example a heterologous nucleic acid molecule such as a plasmid orother low molecular weight nucleic acid, in which case the host cell istypically suitable for replicating the nucleic acid molecule ofinterest. Examples of suitable host cells useful in the presentinvention include, bacteria, algae, and yeast, Specific examples of suchcells include, E. Coli DH5α cells, as well as various other bacterialcell sources, for example the E. Coli strains: DH10b cells, XL1Bluecells, XL2Blue cells, Top10 cells, HB101 cells, and DH12S cells, yeasthost cells from the genera including Saccharomyces, Pichia, andKluveromyces and green alga, for example Chlamydomonas reinhardtii.

“Hybridization” refers to the pairing of complementary polynucleotidesduring an annealing period. The strength of hybridization between twopolynucleotides molecules is impacted by the homology between the twomolecules, stringent conditions involved, the melting temperature of theformed hybrid and the G:C ratio within the polynucleotides. Highstringency conditions include, for example, 42° C., 6×SSC, 0.1% SDS for2 hours.

“Nucleic acid” or “NA” refers to both a deoxyribonucleic acid and aribonucleic acid. As used herein, “nucleic acid sequence” refers to theorder or sequence of deoxyribonucleotides or ribonucleotides along astrand. They may be natural or artificial sequences, and in particulargenomic DNA (gDNA), complementary DNA (cDNA), messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA), hybrid sequences or syntheticor semisynthetic sequences, oligonucleotides which are modified orotherwise. These nucleic acids may be of human, animal, plant, bacterialor viral origin and the like. They may be obtained by any techniqueknown to persons skilled in the art, and in particular by the screeningof libraries, by chemical synthesis or by mixed methods including thechemical or enzymatic modification of sequences obtained by thescreening of libraries. They may be chemically modified, e.g. they maybe pseudonucleic acids (PNA), oligonucleotides modified by variouschemical bonds (for example phosphorothioate or methyl phosphonate), oralternatively oligonucleotides which are functionalized, e.g. which arecoupled with one or more molecules having distinct characteristicproperties. In the case of deoxyribonucleic acids, they may be single-or double-stranded, as well as short oligonucleotides or longersequences. In particular, the nucleic acids advantageously consist ofplasmids, vectors, episomes, expression cassettes and the like. Thesedeoxyribonucleic acids may carry genes of therapeutic interest,sequences for regulating transcription or replication, anti-sensesequences which are modified or otherwise, regions for binding to othercellular components, and the like.

“Oxygen resistant” refers to any measurable decrease in oxygensensitivity in a hydrogenase as compared to a hydrogenase having areference oxygen sensitivity, for example, as compared to a wild typehydrogenase from which an oxygen-resistant hydrogenase enzyme has beenmade.

“Oxygen sensitive” refers to the wild type or reference oxygensensitivity found in a native hydrogenase.

“Protein,” “peptide,” and “polypeptide” are used interchangeably todenote an amino acid polymer or a set of two or more interacting orbound amino acid polymers.

Green Algae and Iron Hydrogenase

Green algae, e.g., Chlamydomonas reinhardtii, cultured under anaerobicconditions synthesize an enzyme known as iron-hydrogenase([Fe]-hydrogenase). As shown in FIG. 1A, several representative[Fe]-hydrogenase enzymes are aligned showing general sequence identityfor the family of proteins. Generally, overall sequence identity for[Fe]-hydrogenase family members is usually at least 45% and sequenceidentity for the H₂-channel between family members is at least 66%.

In general, [Fe]-hydrogenase enzymes characteristically possess acatalytic site consisting of a bimetallic center containing two Fe atoms(2Fe-center), bridged by cysteinyl sulfur to an electron relay [4Fe4S]center (4Fe-center). The iron atoms of the catalytic 2Fe-center arejoined together by a combination of organic, sulfur, and carbon monoxideligands. The chemistry of the [Fe]-hydrogenase catalytic core isreactive with respect to hydrogen, typically possessing very highhydrogen-production rates. However, this same catalytic core is alsohighly sensitive to inactivation by oxygen. As a protective measureagainst inactivation by oxygen or other like molecules, the catalyticcore is typically buried deep within the protein, where access to thecore is limited. As a result, interface of the hydrogenase catalyticsite with surface surroundings is principally limited to a singlechannel, termed the H₂-channel, that directs diffusion of synthesizedhydrogen from the enzyme interior to the external environment. TheH₂-channel is also the primary access route of oxygen to themetallo-catalytic site within hydrogenase enzyme. Reverse diffusion ofthe oxygen from the surface of the enzyme into the H₂-channel and on tothe active site, allows oxygen to bind to the 2Fe-center, inactivatingthe enzyme. Under normal physiologic conditions this represents a fairlynormal inhibitory response for the hydrogenase enzyme, however, underthe artificial conditions of expressing bulk amounts of H₂, this is afairly major limitation.

The present invention provides for the modification of the H₂-channel toreduce oxygen diffusion from the external environment to the enzyme'scatalytic core. The present invention provides modifications to theH₂-channel that act as oxygen filters, preventing or reducing thediffusion of oxygen to the catalytic site within the hydrogenase enzyme.These modifications are at the same time insufficient to limit theability of H₂ to diffuse out of the enzyme through the H₂-channel.Several mechanisms for the reduction of oxygen diffusion to the activesite within the hydrogenase enzyme are provided, including targetedreplacement of residues that line the H₂-channel with bulkier residues,so as to shield the 2Fe-center and/or reduce the diameter of theH₂-channel. In particular, the residues that line the H₂-channel arereplaced with bulkier, hydrophobic residues, for example tryptophan orphenylalanine, so as to shield the 2Fe-center, as well as to reduce thesize or volume of the catalytic site-H₂-channel interface. In addition,modifications to residues on the channel interior that approach anddefine the channel-solvent boundary (see portion 4 of FIG. 3).

In a preferred embodiment of the invention, a process for designing andengineering oxygen-resistant iron-hydrogenases has been developed. Theengineering scheme targets the structure and or environment of theH₂-channel within the target hydrogenase, which is altered to be moreselective in allowing the outward diffusion of hydrogen whilesimultaneously filtering our surface oxygen. Note that size-limiteddiffusion has been successfully used to generate filters for commercialuse in the separation of gases, including the separation of hydrogenfrom oxygen. Menoff T. M., (2000) Proc. Hydrogen Program Review.

The present invention provides host cells for the expression of nucleicacid molecules for encoding an oxygen-resistant iron-hydrogenase, forexample, C. reinhardtii that expresses an oxygen-resistant HydA1 orcyanobacteria that also expresses an oxygen-resistant HydA1. Exampleoxygen-resistant hydrogenases designed and engineered by the method ofthe present invention include V240W, A78W, A244W, A248W, G86W, and L93W.These oxygen-resistant hydrogenase enzymes have the same primarystructure as HydA1 with the exception that A at residue 78 is replacedwith W. Note that other residues besides W, including synthetic andderivatized amino acids, are envisioned for substitution into theH₂-channel, as long as they limit O₂ diffusion through the channel andallow H₂ diffusion out of the channel.

In addition, the invention provides for the bulk photoproduction of H₂using the transformed host cells of the invention.

Identification of the Residues that Form the H₂-Channel of a[Fe]-Hydrogenase

The iron-hydrogenase family of enzymes is a group of enzymes expressedin algae for metabolism of hydrogen. Iron-hydrogenase family membershave been shown to have three distinct motifs that contain highlyconserved residues, including a series of identifiable cysteineresidues. Vignais et al., (2001) FEMS Micro. Rev. 25:455-501. Inparticular, motif 1 has the amino acid sequence PMFTSCCPxW, motif 2 hasan amino acid sequence MPCxxKxxExxR and motif 3 has an amino acidsequence of FxExMACxGxCV. These three motifs have been identified in alliron-hydrogenase family members to date. The cysteine residues have beenshown to either ligate the catalytic [4Fe-4S] center, or bridge the[4Fe-4S] to the [2Fe-2S] center, and there presence within the primarystructure of the enzyme is highly conserved. One of the most studiediron-hydrogenase enzymes is CpI, having its primary, secondary andtertiary structures determined. Peters et al., (1998) Science282:1853-1858. In preferred embodiments, CpI or other like knowniron-hydrogenase enzymes, can be used in the design and engineering ofoxygen-resistant hydrogenases (see below and FIG. 1A for potentialiron-hydrogenase enzymes).

To identify the H₂-channel within a target hydrogenase, i.e., apolypeptide containing motifs 1-3 above, the primary sequence of thetarget hydrogenase must be compared to the primary sequence of a knownhydrogenase. Once the two sequences have been aligned a level ofidentity is determined (see FIGS. 1A and 2). Stothard P., (2000)BioTechniques 28(6) 1102 (hereby incorporated by reference in itsentirety). For purposes of the present invention an overall identity ofapproximately 40%-45% or better should be found for the targethydrogenase. Further, an analysis of the target polypeptide's primarysequence is performed to predict the sequences that share homology withthe H₂-channel forming regions of other known iron-hydrogenases (similarpatterns of residues that have been shown previously to form hydrophobiccavities). Montet et al., (1997) Nat. Struc. Biol. 4:523-526. It shouldbe noted that because the H₂-channel is a conserved domain within allhydrogenases, other non-iron hydrogenase sequences can be used toidentify the target hydrogenase H₂-channel. There should be at least40%-45% identity between the known and unknown sequence between theH₂-channel sequences of the know and unknown hydrogenases. Once theregion within the target polypeptide for the H₂-channel has beenlocated, the channel is modeled into a three-dimensional structureshowing the orientation of residues in relation to the channel andactive site. Guex et al., (1997) Electrophoresis 18:2714-2723. (seebelow) In some embodiments, the analysis is extended to identify theresidues corresponding to the active site within the target hydrogenase.Note that the active site of the target or unknown hydrogenase shouldshare at least 90% homology for motifs 1-3, and in preferred embodimentsshown complete identity with motifs 1-3 (see above). The combination ofprimary and tertiary structures of the target hydrogenase are comparedto evaluate the identification of candidate regions for the finalverification of the hydrogen-channel.

Methods for Designing and Engineering Oxygen-Resistant Iron-Hydrogenases

As noted above, the present invention provides a model for generating atheoretical structure of a target H₂-channel within a target hydrogenaseenzyme. In one embodiment, the theoretical structure is generated byhomology modeling (see above) to the solved structure of other known[Fe]-hydrogenases, for example CpI. (see FIG. 1A). In some embodiments,the homology modeling is limited to the known hydrogenase active siteand H₂-channel, and in other embodiments the homology modeling can belimited to the known hydrogenase H₂-channel sub-domains. A percenthomology of the known hydrogenase (both identity and similarity) can beused to determined residue identity and similarity for the entireenzyme, the active site, the H₂-channel and the H₂-channel sub-domains(see overhead arrows in FIG. 2 and see discussion in previous sectionabove). As such, the present invention provides a known hydrogenasebased homology model that gives a reliable approximation of the targethydrogenase structure and H₂-channel environment. In a preferredembodiment, the known hydrogenase is CpI and the target hydrogenase isHydA1. Homology modeling can be performed using Swiss-model software asdescribed in Guex et al. Guex et al (1997) Electrophoresis 18:2714-2723.Note, however, that other like programs can be used in this aspect, asis known in the art, e.g., Modeller program designed by Marti-Renom etal., (2000) Ann. Rev. Biophy, Biomol. Struct. 29:291-325; EsyPred3Ddesigned by Lambert C. et al., (2002) Bioinformatics 18(9):1250-1256.

Typically, the homology modeling identifies the residues that projectinto the H₂-channel interior of the target hydrogenase. The channelenvironment is often composed of smaller hydrophobic residues, e.g.,glycine, alanine, valine, but can contain phenylalanine and other likeresidues. For example, the H₂-channel of HydA1 contains mostly smallhydrophobic residues with the exception of the larger phenylalanines atpositions 252 and 355 (see FIG. 2, black dotted residues). A secondarystructure is determined from the active site to the enzyme surface usingthe modeled structure above, and distances between side chain atoms ofidentified residues opposed to each other are determined. Guex et al.,Supra An approximate average diameter of the channel over the distancefrom the catalytic site (Fe₂-atom to the H-cluster [2Fe-2S]-center) tothe protein surface is determined (see FIG. 3, 1-4) (typically by usingthe distances between the side chain atoms of opposed residues withinthe channel). In silico mutagenesis is performed on the identifiedhydrogenase H₂-channel structure to identify possible residues that canbe modified to reduce the H₂-channel diameter. Mutagenesis criteriapreferably involve conservative mutation of specific residues, selectionof the lowest energy rotomer and energy minimization of the resultingstructure using GROMOS. van Gunsteren, W. F. et al., (1996) BiomolecularSimulation, The GROMOS96 Manual and User Guide. Vdf HochschulverlagETHZ. Once an energy minimized structure is obtained, the dimensions ofthe target in silico mutagenized hydrogenase channel is determined. Inpreferred embodiments, one or more locations along the H₂-channel isdesigned via conservative mutation to be smaller in diameter than acorresponding non-mutated H₂-channel, typically this reduction is to achannel size of between approximately 5.0 and 2.4 Å in diameter, andpreferably between 3.5 and 2.5 Å, a diameter that either limits oreliminates the ability of oxygen to diffuse through the modifiedH₂-channel. Note that the H₂-channel is in constant flux, as suchdiameter measurements are averages and not meant to be held to a staticstandard. Note that in embodiments of the present invention, more thanone residue can be in silico mutated to design an optimumoxygen-resistant hydrogenase.

In an alternative embodiment, design of oxygen-resistant hydrogenaseenzymes is provided by determining what substitutions/modifications ofresidues within the identified H₂-channel of a target [Fe]-hydrogenasecan be performed to reduce the volume of the H₂-channel. Volumeconsiderations include a reduction in the flow of gasses, i.e., O₂,through the channel in accordance with Stokes Einstein Equation andFick's law.

Designed oxygen-resistant hydrogenases, having a reduced diameterH₂-channel, are genetically engineered and transformed into target hostcells, for example, into C. reinhardtii, and tested for hydrogenaseactivity in the presence of O₂ via a modified Clark electrode or otherknown assay(s). In preferred embodiments, the oxygen-resistanthydrogenase is generated via site-directed mutagenesis. For example, togenerate HydA1 mutants, the HydA1 gene of pA1ExBle can be mutagenized invitro using the Quick Change XL Site-Directed Mutagenesis Kit(Stratagene). Host cells that have incorporated the designed enzymes(having reduced oxygen sensitivity) can be used to photoproduce H₂ in anoxygen containing environment. Note that these host cells can also betreated with mRNA interference to repress the expression of nativehydrogenases, while continuing to allow expression of the inventiveengineered hydrogenase(s).

Steered Molecular Dynamics (SMD)

In one embodiment, the in silico designed oxygen-resistant hydrogenaseenzymes can be further analyzed for changed or reduced oxygen diffusionwithin their H₂-channel by applying SMD via the NAMD program. Kale L. etal., (1999) Computational Physics 151:283; Isralewitz B., (2001) Curr.Opin. Struc. Biol. 11:224. SMD analysis, therefore, providesconfirmation and additional baseline data as to the efficiency of thechannel modifications and their effects on O₂ diffusion within theproposed oxygen-resistant hydrogenase.

Oxygen-Resistant Hydrogenase Polypeptides

Oxygen-resistant hydrogenase enzymes of the invention include allproteins that can be constructed from the in silico mutagenesis methodsdiscussed above. For example, any polypeptide having a predictedreduction in hydrogen-channel diameter or volume, as determined by themethods of the invention, is envisioned to be within the scope of thepresent invention.

In addition, oxygen-resistant hydrogenase enzymes of the inventioninclude isolated polypeptides having an amino acid sequence as shown inFIG. 2 (Cr HydA1), and having one or more substitutions at residuesV240, A78, A244, A248, G86, and L93 (note that substitution bytryptophan and other like amino acids is envisioned, including syntheticor derivatized amino acids) (also included are substitutions shown inTables 1 and 2). The invention includes variants and derivatives ofthese oxygen-resistant [Fe]-hydrogenase enzymes, including fragments,having substantial identity to these amino acid sequences, and thatretain both hydrogenase activity and enhanced tolerance to oxygen (seeExample 3 for assays to determine hydrogenase activity in the presenceof oxygen). In a preferred embodiment, the oxygen-resistant hydrogenaseenzyme is HydA1V240W. Derivatives of the oxygen-resistant hydrogenasesinclude, for example, oxygen-resistant HydA1 enzymes modified bycovalent or aggregative conjugation with other chemical moieties, suchas lipids, acetyl groups, glycosyl groups, and the like.

Oxygen-resistant hydrogenase enzymes of the present invention can befused to heterologous polypeptides to facilitate purification. Manyavailable heterologous peptides allow selective binding of the fusionprotein to a binding partner, for example, 6-His, thioredoxin,hemaglutinin, GST, and the like.

Polypeptide fragments of the modified oxygen-resistant hydrogenaseH₂-channel polypeptide (that include the relevant residue modification)can be used to generate specific anti-oxygen-resistant hydrogenaseantibodies (monoclonal or polyclonal). Generated antibodies can be usedto selectively identify expression of oxygen-resistant hydrogenases orin other known molecular and/or biochemical techniques, for example, inimmunoprecipitation or Western blotting.

Variant oxygen-resistant hydrogenase enzymes include fusion proteinsformed of a oxygen-resistant hydrogenase and a heterologous polypeptide.Preferred heterologous polypeptides include those that facilitatepurification, stability or secretion.

Oxygen-Resistant Hydrogenase Polynucleotides, Vectors and Host Cells

The invention also provides polynucleotide molecules encoding theoxygen-resistant polypeptides of the invention. The polynucleotidemolecules of the invention can be cDNA, chemically synthesized DNA, DNAamplified by PCR, RNA or combinations thereof.

The present invention also provides vectors containing thepolynucleotide molecules of the invention, as well as host cellstransformed with such vectors. Any of the polynucleotide molecules ofthe invention may be contained in a vector, which generally includes aselectable marker, and an origin of replication, for propogation in ahost. The vectors also include suitable transcriptional or translationalregulatory sequences, such as those derived from algae operably linkedto the oxygen-resistant hydrogenase polynucleotide molecule. Examples ofsuch regulatory sequences include transcriptional promoters, operators,enhances, and mRNA binding sites. Nucleotide sequences are operablylinked when the regulatory sequence functionally relates to the DNAencoding the target protein. Thus, a promoter nucleotide sequence isoperably linked to a oxygen-resistant hydrogenase DNA sequence if thepromoter nucleotide sequence directs the transcription of theoxygen-resistant hydrogenase sequence.

Selection of suitable vectors for the cloning of oxygen-resistanthydrogenase polynucleotides of the invention will depend on the hostcell in which the vector will be transformed/expressed. For example, theplasmid pLam91-1 (see FIG. 8) was used to create a 980 bp HydA1 PstIpromoter fragment cloned into a unique PstI site of pLam91-1, creatingthe HydA1 promoter-HydA1 cDNA fusion construct, pA1Ax. The Ble^(r)cassette of pSP108 confers Bleomycin resistance in transformed C.reinhardtii, and was inserted into the Tfi1 site of pA1Ax, creatingpA1AxBle (see FIG. 9). This was particularly useful in the constructionof expression oxygen-resistant [Fe]-hydrogenase vectors for use in greenalgae.

Suitable host cells for expression of target polypeptides of theinvention include green algae, for example C. reinhardtii cells andcyanobacteria, both of which utilize water in growth, which is also asubstrate for the hydrogenase enzymes. Typically, green algae cells aretransformed by a glass bead method as is known in the art. Cellsexhibiting the target selectable marker, for example resistance tobleomycin, are picked and patched onto fresh TAP+Ble plates andre-patched an additional 2-3 times to ensure the isolation of stableintegrates.

H₂ Production

Green algal cultures that express oxygen-resistant hydrogenase of theinvention may be used to photoproduce H₂ in the presence of oxygen. Inone embodiment of the invention, the transformed cells are grown in aphotobioreactor photoautotrophically, photoheterotrophically in TAP, orother like growth media to a concentration of 5-50 μg/ml chlorophyll,and H₂ harvested. Note that in some embodiments, the cells are grownunder selective pressure that ensures that the cells maintain theoxygen-resistant hydrogenase, for example in bleomycin, where theconstruct used to transform the host cell confers the selectivepressure.

In another embodiment, the oxygen-resistant hydrogenase of the inventionmay be transformed into target algae, under the control of theendogenous HydA1 promoter, for nighttime enzyme generation and daytimeH₂-production. See Boichenko et al., (2003) Photoconversion of SolarEnergy, Molecular to Global Photosynthesis: In Press.

It is envisioned that the proceeding discussion on the design,engineering, and construction of oxygen-resistant hydrogenases, as wellas the subsequent transformation of host cells with the designedhydrogenases, can be expanded to any iron hydrogenase known oridentified in the future having the characteristics for iron hydrogenaseenzymes discussed herein.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Computer Modeling of Hydrogenase H₂-Channel forDesign of Oxygen-Resistant Hydrogenase Enzymes

To facilitate the design and engineering of mutant oxygen-resistantHydA1 enzymes, a theoretical structure of HydA1 was generated byhomology modeling to the solved X-ray structure of Clostridiumpasteuraianum [Fe]-hydrogenase, CpI (FIG. 1B). The theoretical model wasgenerated by homology modeling using Swiss-model software as describedby Guex et al. Guex et al., (1997) Electrophoresis 18:2714-2723. Theresulting HydA1 model was subjected to several rounds of energyminimization using GROMOS. An alignment of the HydA1 and CpI amino acidsequences show they share a high degree of homology (45% identity, 58%similarity) within the essential domains, i.e., active site andH₂-channel, that comprise the core region of [Fe]-hydrogenases (see FIG.2). Stothard P., (2000) BioTechniques 28(6) 1102. Note that the degreeof conservation increases for H₂-channel sub-domains, where the twoproteins share 62% identity and 92% similarity (FIG. 2, overheadarrows). This high level of sequence identity/similarity shows that theCpI-based HydA1 homology model provides a reasonable approximation ofthe HydA1 structure and the H₂-channel environment.

The detailed study of the HydA1H₂-channel structure was performed, atleast partly, to identify residues that project into the H₂-channelinterior. In general, the channel environment was primarily composed ofsmaller hydrophobic residues, e.g., glycine, alanine, valine, with theexception of the larger phenylalanines at positions 252 and 355 (FIG. 2,black dotted residues). The secondary structure of the H₂-channel wasorganized into two α-helices and two β-sheets, which extend from theactive site to the enzyme surface. The distance between side chain atomsof residues that oppose each other were measured to approximate theaverage channel diameter over the distance from the catalytic site(Fe₂-atom of the H-cluster [2Fe-2S]-center) to the protein surface (1 to4, FIG. 3). The channel measured 3.85 to 7.44 Å in diameter over adistance of 24 to 27 Å, making the channel diameter greater than theeffective diameters of both H₂ (2.8 Å) and O₂ (3.5 Å). As a result, thepredicted size of the HydA1 H₂-channel is sufficient to function in H2diffusion from the active site to the surface, but it is also largeenough to allow for the inward diffusion of the inhibitor O₂.

These results suggest that engineering O₂ tolerance into HydA1 might beaccomplished by altering the residues that line the interior of thechannel so as to reduce the diameter of the channel and thereby limit O₂diffusion to the active site. The potential to reduce the diameter ofthe channel via residue substitution was initially tested in silico bymutating the H₂-channel of the HydA1 model. The mutagenesis criteriainvolved conservative mutation, i.e., hydrophobic→hydrophobic, ofspecific residues, selection of the lowest energy rotomer, and energyminimization of the resulting structure using GROMOS. Once anenergy-minimized structure was obtained, the dimensions of its channelwas determined. Several of the channel residues proved to be unameanableto mutation and were left unchanged, i.e., I82, L89, F252 and F355,i.e., the Guex program determined that changes at these locations wouldprovide only minimal (non-significant) change to the H₂-channeldiameter/volume. However, promising mutants were generated fromalteration of several residues that were spaced over the entire lengthof the channel (see FIG. 2 and Table 1). Substantial reductions ofchannel diameter were obtained by mutating residues A78 and V240(proximal to active site); A244, A248 and G86 (mid-channel); and L93(protein-solvent boundary, distal to active site) to bulkier amino acids(Table 1 and FIG. 3). The individual mutations listed in Table 1 causedreductions in diameter that ranged from 0.5 to 1.90 Å (Table 2). TheHydA1 mutant that combined the A248I and L93F mutations located at thechannel-solvent boundary (FIG. 3, zone 4) showed an average decrease insize from 5.21 to 3.34 Å, less than the effective diameter of O₂ (3.5Å). When the mutations listed in Table 2 were combined into a singleHydA1 mutant, the average overall channel diameter was reduced from anaverage 5.71 to an average of 4.31 Å (Table 2), noting however thatthere are several locations along the H₂-channel with reductions in thediameter at or near the average diameter of O₂.

TABLE 1 Predicted Effects of Selected HydA1 H2-Channel Mutations onChannel Environment Mutation Location Effects A781 Adjacent to Fe₂-atom,Bulkier isoleucine side across from V240 chain projects closer to V240,and the [2Fe—2S]- center Fe₂ atom. V240W Adjacent to A78, above Bulkiertryptophan side Fe₂-atom. chain reduces distance to A78, and partiallyshields Fe₂-atom. A244L Mid-channel, opposes I82 Bulkier leucine sidechain projects further into channel. G86I Mid-channel, opposes A248Isoleucine side chain adds bulk, and projects into channel. A248IMid-channel, near surface, Isoleucine extends further opposes G86 andL89 into channel, adds more bulk to hydrophobic surface. L93FChannel-Surface boundary Narrows the channel opening at protein surface-solvent boundary

TABLE 2 Distances Between Channel Determinants In HydA1 and HydA1Mutants Based on Modeling Studies Distances (Å) Average Zone Size (Å)Determinant HydA1 HydA1 HydA1 HydA1 Zone^(a) Pairs^(b) wild typemutant^(c) wild type mutant^(c) 1 A(I)78::Fe₂ 6.25 4.66 6.90 5.04V(W)240::Fe₂ 6.14 5.02 V(W)240::A(I)78 7.43 5.43 2 A(L)244::I82 4.503.90 5.30 4.87 F355::G(I)86 6.86 5.80 3 G(I)86::A(I)248 4.38 3.54 5.423.92 G(I)86::T247 6.01 4.39 A(I)248::L89 3.85 3.26 A(I)248::F355 7.444.54 4 L90::A(I)248 6.11 3.67 5.21 3.34 L(F)93::F252 4.31 3.01 ^(a)Thelocations of H2-channel zones are identified in FIG. 3. ^(b)Determinantsare identified as wild-type, with corresponding mutations inparentheses. ^(c)Measurements are averages of a HydA1 mutant possessingall the identified mutations within the designated zone.

The above results indicate that modeling of the HydA1 structure hasrevealed a hydrophobic channel extending from the active site to theenzyme surface. This channel would appear to be conserved in other[Fe]-hydrogenases. The channel's secondary structure is mainlyα-helical, which suggests that the channel domain is fairly rigid.Perhaps the rigidity of the channel structure helps to prevent itscollapse during folding. Volbeda et al., (2002) Int. J. Hyd. Energy27:1449-1461. Rigidity would also be expected to contribute toconformational stability of the channel in the folded protein, and astatic model should give reasonable approximations of shape and size.Our measurements of the HydA1 channel demonstrate that it is sufficientin diameter not only to allow for diffusion of the product H₂ but alsothe larger-sized inhibitors O₂ and CO. Since enzyme inhibition occursquickly (minutes), following exposure of O₂ (Happe et al., (1994) Eur.J. Biochem. 222:769-774), the channel would not appear to be highlyrestrictive to inhibitor diffusion, which is in agreement with ouranalysis. This data illustrates the utility of the present invention forengineering O₂-resistant, [Fe]-hydrogenase by manipulation of residueswithin the conserved H₂ channel. This modeling approach can be used inenzymes that have one channel or multiple channels to reduce inhibitoraccess to an enzyme active site.

Example 2 C. reinhardtii can be Transformed with HydA1 H₂-ChannelMutants

To test the ability of the predicted HydA1H₂-channel mutants forlimiting O₂ inhibition, an algal HydA1 expression system was createdusing the HydA1 endogenous promoter. From the modeling discussed inExample 1, the V240W mutation was selected for further examination. Invivo expression of the V240W mutant was performed and further testing ofthe mutant for O₂ resistance hydrogenase activity performed. Note thatthe V240W mutation is predicted to cause a constriction of the channelnear the active site (see FIG. 4). In addition, the tryptophan projectsover the Fe₂-atom, partially shielding it from the channel domain.

The Chlamydomonas reinhardtii strain cc849 (cw10, mt-) was used as thewild type parent strain throughout the remainder of this Example. Growthof liquid cultures were performed photoheterotrophically in TAP medium(Harris E, (1989) The Chlamydomonas Source Book, Academic Press, NewYork) with a continuous stream of 5% CO₂ under cool-white fluorescentlight (150 μE/m⁻²/s⁻¹ PAR). Growth on solid medium was performed on TAPagar plates (TAP medium with 1.4% w/v agar). Note that when selection ofBleomycin resistance was performed, solid TAP medium was supplementedwith 10 μg/ml Zeocin (Invitrogen).

A plasmid construct pLam91-1, containing the HydA1 cDNA and3′-terminator regions cloned into the EcoRI-XhoI sites of pBluescriptSK, was used to generate an algal HydA1 expression construct. A 980 bpHydA1 PstI promoter fragment was cloned into the unique PstI site ofLam91-1, creating the HydA1 promoter-HydA1 cDNA fusion construct, pA1Ex.The Ble^(r) cassette of pSP108 that confers Bleomycin resistance intransformed C. reinhardtii (Stevens et al., (1996) Mol Gen Genet.251:23-30) was inserted into the TfiI site of pA1Ex, creating pA1ExBle.

Site-directed mutagenesis was performed on HydA1 to generate HydA1mutants for expression in C. reinhardtii. The HydA1 gene pA1ExBle wasmutagenized in vitro using the Quick Change XL Site-Directed MutagenesisKit of Stratagene. Oligonucleotides (Integrated DNA Technologies) usedfor mutagenesis were designed based on the kit requirements. MutantpA1ExBle constructs were sequenced to confirm the presence of individualmutations. The HydA1 mutant, V240W, contains a valine to tryptophansubstitution at amino acid position 240 of the mature protein.

C. reinhardtii cells were next transformed by the glass bead method asis known in the art (see also Harris E) using 10 μg of linearizedpA1ExBleV240W DNA. Following transformation, cells were culturedovernight in 2 ml of TAP medium to allow for cell recovery andphenotypic expression of Ble^(r). Transformed cells were harvested bycentrifugation (2000×g, 5 minutes), resuspended in 1.5 ml TAP soft agar(TAP with 0.8% w/v agar) and spread onto TAP+Ble agar plates. Plateswere incubated in the light for a period of 1-2 weeks and Ble^(r)colonies picked. Resistant colonies were patched onto fresh TAP+Bleplates, and re-patched an additional 2-3 times to ensure the isolationof stable integrates.

To ensure that the HydA1 cDNA genomic insert having the V240W mutationwas present in the transformed C. reinhardtii, PCR and sequencing wasperformed on Ble^(r) transformants. Total genomic DNA was isolated fromindividual Ble^(r) transformants using the Plant Genomic Kit (Qiagen). Atotal of 0.5 to 1.0 μg of purified genomic DNA was digested with eitherSacI or EcoRI and used as template in a PCR reaction consisting of theHydA1 internal primers (5′-CACGCTGTTTGGCATCGACCTGACCATCATG-3′ and5′-GCCACGGCCACGCGGAATGTGATGCCGCCCC-3′), 1 unit KOD HotStart polymerase(Novagen), 10 mM MgSO4, 25 mM of each dNTP, 2% DMSO (v/v), and water toa total volume of 50 μl. The presence of a HydA1 cDNA genomic insertresults in an additional 780 bp HydA1 cDNA product together with the1120 bp HydA1 genomic product. PCR reactions were run on 1×TAE agarosegels (1.25% agarose w/v), stained with ethidium bromide, andphotographed (not shown). The 780 bp band, corresponding to the HydA1cDNA insert, was purified and sequenced to confirm the presence of V240Wmutation. Two Ble^(r) C. reinhardtii clones, mt18 and mt28, were shownto possess the HydA1V240W construct (see FIG. 5).

Example 3 Green Alga, C. reinhardtii, Transformed with Oxygen-ResistantHydrogenases are Effective in the Bulk Production of H₂

The O₂-sensitivity of [Fe]-hydrogenase activity in strains mt18 and mt28carrying the HydA1V240W mutation was tested in either whole cells orwhole cell extracts of anaerobically induced cultures. Hydrogenaseactivities were measured as H₂ gas photoproduction by whole cells aspreviously described. Ghirardi et al., (1997) App. Biochem. Biotech.63-65:141-151; Flynn et al., (2002) Int. J. Hyd Energy 27:1421-1430.Briefly, cells were grown photoheterotrophically in TAP to aconcentration of 15-20 μg/ml chlorophyll, harvested and resuspended at200 μg/ml chlorophyll in phosphate induction buffer. Ghirardi et al.Clark electrode measurement of O2-resistant hydrogenase activity wasperformed by adjusting the O₂ concentration in the electrode chamber toa set level between 0% and 4%. Once the O₂ level had stabilized, astream of Ar gas was passed over the chamber to maintain a constant O2concentration. A 0.2 ml sample of induced cell suspension was injectedinto the chamber, and the cells kept in the dark for a two minuteperiod. Light dependent H₂-photoproduction activity was then induced byillumination.

In addition, to measure hydrogenase activity directly, reduced methylviologen (MV) was used as an artificial electron donor for H₂ productionby solubilizing whole cells as previously described. Flynn et al.Tolerance to O₂ was measured by incubating 1 ml of induced cells in adark, sealed glass bottle and injecting O₂ to achieve a final atmosphereof 1 to 4% (v/v). Samples were incubated for two minutes then purgedwith Ar gas for five minutes. A 1 ml mixture of reduced MV and TritonX-100 in a phosphate buffer was added, samples were mixed for three tofive minutes, and 0.1 ml of 100 mM reduced Na-dithionite injected tostart the reaction. The reaction mixtures were incubated for 30 minutesat room temperature with stirring, and reactions were stopped by theaddition of 0.1 ml 20% trichloroacetic acid (TCA). The hydrogen contentof a 0.2 ml headspace sample was measured by gas chromatograph. Threeseparate headspace samples were assayed, and the values were averaged toattain final hydrogen-production rates.

As shown in Table 3, all three strains, cc849, mt18, and mt28, exhibitedsimilar levels of hydrogenase activity (rate of H₂ photoproduction)under completely anaerobic conditions. Note that as has been shown inprevious studies (Ghirardi et al, supra; Flynn et al, supra),pretreatment of induced wild type cells with O₂ is sufficient to cause asignificant decline in H2 production rate (FIG. 6, white bars). Wheninduced wild-type cells were pre-treated with O2 at a concentration of1.7 to 3.5%, the H2 photoproduction rate declined by 90 to 100%respectively. However, the exposure of mt18 or mt28 induced cells tosimilar O2 treatments showed H2 photoproduction activity had significantresistance to inactivation. After exposure to 1.7 to 2.2% O₂concentrations, the H₂ photoproduction rates remained 3.8 to 7 foldhigher in mt18, and 3.2 to 13 fold higher in mt28 compared to activitiesin wild-type cells under identical conditions (see FIG. 6). At 3.5% O₂treatment, the H2 photoproduction rates in both mt18 and mt 28 were low,but detectable, whereas residual activity in wild-type cells wasundetectable (FIG. 6).

TABLE 3 Hydrogenase Activity By the Clark Electrode Assay H₂Photoproduction Rate Strain (μmol H2/mg chl⁻¹/h⁻¹) cc849 10.4 mt18 14.1mt28 10.7

The light-induced production of hydrogen by whole cells is a metabolicprocess and depends on many electron transfer steps. Zhang et al.,(2000) Trends Biotech. 18(12):506-511; Melis et al., (2001) PlantPhysiol. 127:740-748; Melis et al., (2000) Plant Physiol. 122:127-135. Amore direct measurement of hydrogenase activity can be accomplished insolubilized whole cells using reduced MV (Mv_(red)) as electron donorfor H2 gas production by hydrogenase in the dark. Under completelyanaerobic conditions, the Mv_(red)→H₂ reaction rates were similar invalue for either induced wild-type or mutant cells (see Table 4). Asshown in FIG. 7, a two-minute exposure of induced wild-type to variousO₂ concentrations caused hydrogenase activity to decline. After exposureof O₂ concentrations of 1% to 4% hydrogenase activities in wild-typecells decreased to between 10 and 1.5% respectively (FIG. 7), similar tothe results shown in FIG. 6. In comparison, both mt16 and mt28containing the HydA1V240W construct exhibited significant levels of O₂resistant hydrogenase activity (see FIG. 7). Exposure of mt18 to O₂ at1% to 4% concentration caused hydrogenase activities to decline by 76%to 96%, whereas mt28 activities declined only 12% to 76% (FIG. 7). As aresult, mt18 hydrogenase activities were 2- to 3-fold higher, and mt28activities 8- to 15-fold higher than activities in wild-type cells afterexposure to similar O₂ treatments.

TABLE 4 Hydrogenase Activity By the Methyl Viologen Assay H2Photoproduction Rate Strain (μmol H2/mg chl⁻¹/h⁻¹) cc849 31.3 mt18 32.9mt28 35.5

This Example illustrated the utility of modeling residue substitutionswithin the H₂-channel to constrict the channel from O₂ passage to the[2Fe-2S]-center. In particular, the Example illustrated thatsubstitution of tryptophan for valine at position 240 of HydA1 caused anincrease tolerance to O₂ in the mutant hydrogenase. The difference inthe structure change made to HydA1V240W and the effects of that changeare similar to the observed differences in structure and O₂-resistanceof H₂-sensing [NiFe]-hydrogenases compared to catalytic[NiFe]-hydrogenases. Volbeda et al., (2002) Int. J. Hyd. Energy27:1449-1461; Bernhard et al., (2001) 276:15592-15597. Active-siteproximal channel residues of O₂-resistant, H₂-sensing[NiFe]-hydrogenases contain the bulky, hydrophobic amino acidsisoleucine and phenylalanine. Identical positions in the O₂-sensitive,catalytic [NiFe]-hydrogenases encode the smaller-sized residues valineand leucine respectively. The difference in amino acid composition issuggested to result in the shielding of the [NiFe]-cluster andconstriction of the channel. Volbeda supra and Bernhard supra.

The invention has been described with reference to specific examples.These examples are not meant to limit the invention in any way. It isunderstood for purposes of this disclosure, that various changes andmodifications may be made to the invention that are well within thescope of the invention. Numerous other changes may be made which willreadily suggest themselves to those skilled in the art and which areencompassed in the spirit of the invention disclosed herein and asdefined in the appended claims.

This specification contains numerous citations to patents andpublications. Each is hereby incorporated by reference in their entiretyfor all purposes.

1. An oxygen-resistant iron hydrogenase derived from an oxygen sensitiveiron hydrogenase by the substitution of one or more amino acid residueswithin the hydrogen channel of the oxygen-sensitive iron hydrogenase. 2.The oxygen-resistant iron hydrogenase of claim 1 wherein saidsubstitution is made to one or more amino acid residues selected fromthe group consisting of residues 78, 240, 244, 86, 248, 247, 82, 89,355, 93 and 252 of HydA1 iron hydrogenase.
 3. The oxygen-resistant ironhydrogenase of claim 1 wherein the substituted amino acid which is at aresidue within said hydrogen channel has a side chain volume which islarger than the side chain volume of the amino acid at the same residuein said oxygen-sensitive iron hydrogenases.
 4. A nucleic acid encodingthe oxygen-resistant iron hydrogenase of claim
 1. 5. A vector comprisingthe nucleic acid of claim
 4. 6. Host cell transformed with the vector ofclaim
 5. 7. The host cell of claim 6 wherein the host cell is greenalgae.
 8. A process for producing hydrogen in green algae comprisingculturing green algae cells transformed with the vector of claim 5 underconditions wherein the nucleic acid encoding said oxygen-resistant ironhydrogenase is expressed.
 9. The process of claim 8 wherein said greenalgae is Chlamydomonas reinhardtii.
 10. A method for making a nucleicacid encoding an oxygen-resistant iron hydrogenase comprising: comparingthe sequence of a first oxygen-sensitive iron hydrogenase having knownamino acid residues that form a hydrogen channel in said firsthydrogenase with a second iron hydrogenase to identify residues thatform a hydrogen channel in said second hydrogenase, and forming anucleic acid encoding a derivative of said iron hydrogenase wherein oneor more amino acid residues within said hydrogen channel are modified toreduce the oxygen sensitivity of said second hydrogenase.
 11. The methodof claim 10 wherein the crystal structure of said first hydrogenase isknown and said comparing is by in silico homology modeling.
 12. Themethod of claim 10 wherein said first hydrogenase is CpI and said secondhydrogenase is HydA1.
 13. A process for making a green algae capable ofhydrogen production in the presence of oxygen comprising transforming agreen algae with the vector of claim
 5. 14. A method of making anoxygen-resistant iron-hydrogenase comprising: determining the diameterof an H₂-channel defined by a set of amino acid residues in anoxygen-sensitive iron hydrogenase by measuring the distance between theside chains of one or more of the amino acid residues of said set toidentity one or more diameter determining amino acid residues; andmodifying one or more of said diameter determining residues in saidoxygen-sensitive iron hydrogenase to reduce the effective diameter ofsaid H₂-channel to form an oxygen-resistant iron hydrogenase, wherebythe diffusion of oxygen within said modified H₂-channel in saidoxygen-resistant iron hydrogenase is reduced as compared to thediffusion of oxygen in said H₂-channel of said oxygen-sensitive ironhydrogenase.
 15. The method of claim 14, wherein said H₂-channelresidues are identified by homology modeling to a known x-ray structureof one or more known hydrogenases.
 16. The method of claim 15, whereinsubstitution at one or more of said diameter determining residues ofsaid H₂-channel is made in the form of an in silico molecule wherein themost energentically favorable rotamer for the substituted side chain isused to minimize the energy of at least the H₂-channel of said molecule.17. The method of claim 16 wherein GROMOS is used to compute saidminimized energy of said molecule.
 18. The method of claim 16 whereinthe diameter of said H₂-channel in said energy minimized molecule iscompared to the diameter of said H₂-channel prior to in silicomodification as a basis for selection of one or more mutations to beformed in said oxygen-sensitive iron hydrogenase protein.